The invention relates in general to episomal vectors.
In lower organisms, such as prokaryotes and budding yeast, replication origins contain welldefined cis-sequences called xe2x80x9creplicatorsxe2x80x9d and interaction of these sequences with a specific initiator protein complex leads to the initiation of DNA synthesis in these cells (Jacob et al., 1963; Stillman, 1994 and references therein; DePamphilis, 1993). Extrachromosomal replicators, generally, in addition to their origin function, encode functions that assure equal distribution of replicated molecules (i.e., partitioning) between daughter cells at cell division. For prokaryotic plasmids these partitioning functions are well studied and can be provided by several different mechanisms in bacterial cells (Nordstrxc3x6m, 1990). In higher organisms, less is known about mechanisms for partitioning of extrachromosomal replicators. For artificial plasmids in yeast, chromosomal centromeres can provide this function. In metazoan cells, one well studied example of a stable extrachromosomal replicator existsxe2x80x94the latent origin oriP from Epstein-Barr Virus (EBV). The maintenance function of EBV requires the viral replication factor EBNA-1 and a series of binding sites for EBNA-1 termed the family of repeats (FR). A model that has been suggested for the function of the EBNA-1/FR combination is that EBNA-1 bound to FR provides physical retention of the oriP plasmids in the cell nucleus (Krysan et al., 1989).
Papillomaviruses are also capable of stable extrachromosomal replication. Infection and transformation of the cells by papillomaviruses follows single hit kinetics. (Dvoretzky et al., 1980). Papillomavirus genomes are maintained as multicopy nuclear plasmids in transformed cells. The viral life-cycle can be viewed as three stages (Botchan et al., 1986). First, following initial entry, the papillomaviral genome is amplified in the cell nucleus, i.e., viral DNA is synthesized faster than chromosomal DNA and the copy-number is increased. The second stage represents maintenance of the viral DNA at a constant copy-number and latent phase of the viral infection is established. During the third, vegetative, stage of the viral life-cycle viral DNA amplification is initiated again, late proteins are synthesized and viral particles are assembled.
The E1 and E2 proteins are the only viral factors required for initiation of papillomavirus DNA replication (Ustav and Stenlund 1991; Ustav et al., 1991; Yang et al., 1991; Chiang et al., 1992; Kuo et al., 1994). A similar, if not identical, set of cellular replication factors and enzymes, in addition to viral initiator proteins, is utilized by SV40 (Tsurimoto et al., 1990; Weinberg et al., 1990) and BPV-1 (Muller et al., 1994) at the origin of replication to initiate DNA synthesis. Analysis of the essential cis-sequences shows that the BPV-1 minimal origin (Ustav et al., 1993) resembles a typical eukaryotic origin of replication (DePamphilis, 1993) and it has been suggested that this similarity could also be extended to the mechanisms of replication of all papovaviruses (Nallaseth and DePamphilis, 1994; Bonne-Andrea et al., 1995). However, the ability of the papillomaviruses to persist as plasmids distinguishes papillomaviruses from other papovaviruses. It has been known for more than 10 years that BPV-1 replicates in transformed cells as a multicopy nuclear plasmid, which can persist in the tissue culture cells over long periods of time (Law et al., 1981). This indicates that papillomaviruses have efficient mechanisms for segregation, i.e., control of copy-number and partitioning, in the transformed cells.
The role of viral factors, cis-acting sequences and cellular factors in long-term persistence of papillomaviruses, which relates to the segregation functions of viral DNA, is not clearly understood. That is, the regions of the viral genome which specify copy number are not identified in the prior art; nor are the regions of the viral genome which participate with the host cell to ensure proper segregation of the viral genome during partitioning. Much more is understood with respect to the initial amplification stage of the papillomavirus life-cycle.
Bovine Papillomavirus (13BPV) and Human Papillomaviruses (HPVs) persist as stably maintained plasmids in mammalian cells. Transient assays, i.e., on the order of several hours to 3-4 days, have been used to define the minimal origin of replication (MO) which is required for transient replication in BPV (Ustav et al., EMBO J, 10, 4231-4329, 1991) and for several HPV subtypes. Two trans-acting factors encoded by BPV and HPVs, namely E1 and E2, have been identified in transient assays which are necessary to mediate replication in many cell types via MO (Ustav et al., EMBO J., 10, 449-457 (1991); Ustav et al., EMBO J, 10, 4231-4329, (1991); Ustav et al., PNAS, 90, 898-902 (1993).) E1 and E2 from BPV will replicate via the BPV MO and via the MO of many HPV subtypes. (Chiang et al., PNAS, 89, 5799-5803 (1992). E1 and E2 from HPV will replicate via the BPV MO and via the MO of many HPV subtypes. (Chiang et al., PNAS, 89, 5799-5803 (1992). Replication of plasmids containing the above elements is high level but transient in eukaryotic cells. Plasmid loss is rapid in the presence and absence of selective pressure.
The papillomavirus life cycle has been the subject of much research. Different portions of the viral genome have been tested in short-term, i.e., transient, transcription or replication assays. See, for example, Szymanski et al., 1991, Jour. Virol. 11:5710; Vande Pol et al., 1990, Jour. Virol 64:5420; Sowden et al., 1989, Nucl. Acids Res. 17:2959; Stenlund, 1987, Science 236:1666; Sedman et al., 1995, Eur. Jour. Mol. Biol. 14:6218; Haugen et al., 1988, Eur. Jour. Mol. Biol. 7:4245; and Kuo et al., 1994, Jour. Biol. Chem. 269:24058.
The BPV 69% transforming region has been used to introduce the rat preproinsulin gene into mouse cells (Sarver et al., 1981, Mol. Cell. Biol. 6:486).
The PMS1 and PMS2 regions of BPV have been reported to xe2x80x9cindependently supportxe2x80x9d extrachromosomal replication of the Tn5 neomycin gene in cells that provide viral factors in trans. PMS-1 (plasmid maintenance sequence-1) is localized within a 521 bp region mapping at positions 6945-7476 of the BPV genome, and PMS-2 has been localized to a 140 bp region within the putative open reading frame for the E1 protein, which maps at positions 1515-1655 of the BPV genome. It has been reported that recombinant plasmids carrying either of the PMS elements are unrearranged and stably maintained at a constant copy number. In addition, E1, E6 and E7 are identified as candidate factors for trans regulation of the plasmid state. See Lusky et al., 1984, Cell 36:391, and Lusky et al., 1986, Jour. Virol. 11:729.
Woo et al., W094/12629 report a vector containing a papilloma virus origin of replication, the xe2x80x9cvector maintenance sequencexe2x80x9d described in Lusky et al., 1984, supra, a therapeutic nucleic acid, and an E2 gene sequence or an E1/E2 chimeric gene. Woo et al. suggest that such a vector may be tested for stable episomal maintenance over a period of 2-30 days post-transfection. The xe2x80x9cvector maintenance sequencexe2x80x9d of Lusky et al., 1984, which is described in Woo et al., is shown herein not to be capable of providing long-term vector persistence.
Mutations in the E2 gene have a pleiotropic effect on viral gene functions, including oncogenic transformation. These effects may be the result of the requirement for E2 expression to regulate viral transcription (see DiMaio and Neary, 1989, The Genetics of bovine papillomavirus type 1 papillomaviruses and human cancer. (Ed. N. Pfister), CRC Press, Boca Raton, Fla.). The BPV-1 E2 protein has been shown to activate viral enhancers in trans (Spalholz et al., 1985, Cell 42:183). The E2 open reading frame has been shown to encode a site-specific DNA binding protein that can bind to several sites within the E2 responsive enhancers 1 and 2 (Androphy et al., 1987, Nature 325:70; Moskaluk et al., 1987, Proc. Nat. Aca. Sci. 84:1215). E2 recognition sites that have been studied to date include the sequence motif ACCN6GGT, (SEQ ID NO: 1) where N is any nucleotide (Hawley-Nelson et al., 1988, Eur. Jour. Mol. Biol. 7:525; Hirochika et al., 1988, Genes Dev. 2:54; McBride, 1988, Eur. Jour. Mol. Biol. 7:553; Moskaluk et al., 1988a, Prc. Nat. Aca. Sci. 85:1826), and it is suggested that E2 binds this palindrome as a dimer (Dostani et al., 1988, Eur. Mol. Biol. Org. Jour. 7:3 807; McBride et al., 1989, Proc. Nat. Aca. Sci. 86:510). Not all of these sites appear to bind E2 with the same strength. Sites having the motif ACCGN4CGGT (SEQ ID NO: 2) appear to bind better than sites that deviate in the fourth and ninth bases (Hawley-Nelson et al., 1988, supra; Moskaluk et al., 1988b, supra). It also appears that some of the target sites for the protein have different capabilities for activation in vivo (Harrison et al., 1987, Nucl. Acids. Res. 15:10267; Haugen et al., 1987, Eur. Jour. Mol. Biol. 6:145; Spalholz et al., 1987, Jour. Virol. 61:2128). Li et al. (1989, Genes and Develop. 510) analyze 17 E2 binding sites in the BPV-1 genome and show that affinities for E2 vary over a 300-fold range. Li et al. also find that the presence of the conserved consensus ACCGN4CGGT (SEQ ID NO: 2) did not necessarily guarantee that the binding site would be stronger than one with a deviant base, and suggest that the presence of this palindrome is not a sufficient parameter for predicting the strength of a binding site.
A truncated form of E2 protein exists which is defective in transcriptional activation and competent in viral replication.
Dowhanick et al., 1995, Jour. Virol. 69:7791, describe an E2 deletion mutant containing residues 1-218 of the protein which is said to retain a DNA replication function, but is defective in transcriptional trans-activation. Also described are several E2 point mutants (331 and 344) which are defective in both transcriptional transactivation and DNA binding.
In addition, subsequent to Applicant""s disclosure of E2 point mutants which are defective in transcriptional activation and replication competent in the subject priority document, which E2 mutants are also described in Abroi et al., 1996, Jour. Virol. 70:6169, additional similar, if not identical in some instances, E2 point mutants have been identified. Ferguson and Botchan, 1996, Jour. Virol. 70:4193, describe mutations at amino acids 73 and 74 which retained replication function but failed to activate transcriptionxe2x80x9d. Sakai et al., 1996, Jour. Virol. 70:1602, describe three point mutants (R37A, 173A, and W92A) in BPV defective for transcriptional activation but retaining wild type DNA replication activity in transient assays.
One object of the invention is to provide a recombinant vector which, by virtue of the sequences it contains, is stably maintained and thus persists long-term in mammalian cells.
Another object of the invention is to provide a recombinant episomal vector which is stabilized via regulatory sequences which are contained within a relatively small amount of DNA.
Another object of the invention is to provide a cis-acting element which confers long-term stability to a transiently replicating eukaryotic episomal plasmid.
Yet another object of the invention is to provide an episomal genetic element which replicates independently of the host cell chromosomal DNA, and is therefore not dependent upon regulatory control of replication by the host cell genome.
Another object of the invention is to provide stable and reliable plasmid copy number in both G1 and G2 stages of the cell cycle.
Yet another object of the invention is to provide a recombinant vector which is stably maintained at a constant copy number for multiple cell generations.
Another object of the invention is to provide a recombinant vector which is able to persist over a long time period in eukaryotic, particularly mammalian cells, from which can be expressed a therapeutic, prophylactic, or marker gene.
Another object of the invention is to provide a recombinant vector which is not restricted as to its ability to be maintained in a given cell type, but which is stably maintained in any one of numerous diverse mammalian cell types.
Another object of the invention is to provide a recombinant vector containing sequences of viral origin which do not confer oncogenic properties to the transfected host cell, and is therefore safe to use in humans.
The invention is based on the discovery of a vector system which permits long-term persistence in episomal form in a mammalian cell, and in particular to the discovery of a minichromosomal maintenance element, which element confers stable persistence of extrachromosomal (i.e., episomal) DNA in mammalian host cells.
The invention encompasses a method of obtaining long-term stable production of a gene product of interest in a host cell, comprising providing a host cell containing a vector comprising (A) a minimal origin of replication of a papilloma virus, (B) a minichromosomal maintenance element of a papilloma virus, and (C) a gene encoding the gene product, wherein the vector, when present in a mammalian host cell, persists in the cell for at least about 50 cell generations in dividing cells or for at least about 8 weeks in non-dividing cells under nonselective conditions without an appreciable loss of copy number.
The invention also encompasses a method of obtaining long-term stable production of a gene product of interest in a host cell, comprising providing a host cell containing a vector comprising papillomavirus sequences consisting essentially of (A) a papillomavirus E2 gene, (B) a minimal origin of replication of a papilloma virus,(C) a minichromosomal maintenance element of a papilloma virus, and (D) a gene encoding the gene product, wherein the vector persists in the cell for at least about 50 cell generations in dividing cells or for at least about 8 weeks in non-dividing cells under nonselective conditions without an appreciable loss of copy number.
The invention also encompasses a method of obtaining long-term stable production of a gene product of interest in a host cell, comprising providing a host cell containing a pair of vectors comprising (I) a first vector comprising papillomavirus sequences consisting essentially of (A) a papillomavirus E2 gene, (B) a minimal origin of replication of a papilloma virus, and (C) a minichromosomal maintenance element of a papilloma virus, and (II) a second vector comprising papillomavirus sequences consisting essentially of (A) a gene encoding the gene product, (B) a minimal origin of replication of a papilloma virus, and (C) a minichromosomal maintenance element of a papilloma virus, wherein the vector persists in the cell for at least about 50 cell generations in dividing cells or for at least about 8 weeks in non-dividing cells under nonselective conditions without an appreciable loss of copy number.
The invention also encompasses use of a recombinant vector for obtaining long term stable maintenance of erogenous DNA in a eukaryotic host cell wherein the recombinant vector comprises: a minimal origin of replication of a papillomavirus; a minichromosomal maintenance element of a papillomavirus; and a heterologous DNA sequence encoding an expressible gene.
Preferably, the time period over which the vector persists in the host cell under nonselective conditions without an appreciable loss of copy number is 6 weeks, and most preferably 8 weeks or 12 weeks or longer, or in terms of cell generations, 100 or 120 cell generations or longer.
According to the claimed methods, long-term persistent vectors will include one in which the minichromosomal maintenance element consists essentially of the region of BPV mapping to positions 7590 to 7673; or wherein the minichromosomal maintenance element comprises (BPV E2 binding sites 6, 7 and 8) x, wherein x is 3 to 6 or wherein the minichromosomal maintenance element comprises at least 2 of the 3 E2 binding sites 6, 7 and 8.
The invention therefore also encompasses a recombinant vector for stable long-term persistence of erogenous DNA in a mammalian host cell, the vector comprising a minimal origin (MO) of replication of a papillomavirus, a minichromosomal maintenance element (MME) of a papillomavirus, and a gene encoding a gene product of interest, wherein the vector is defined hereinbelow (1-4).
1. The vector comprising papilloma virus sequences consisting essentially of (A) a minimal origin of replication of a papilloma virus, (B) a minichromosomal maintenance element of a papilloma virus consisting essentially of at least two of the three E2 binding sites 6, 7, and 8, wherein the region of the vector comprising the minimal origin of replication and minichromosomal maintenance element consists of a DNA sequence different from the natural papilloma virus sequence, and wherein the vector, when present in a mammalian host cell which expresses E1 and E2, persists in the cell for at least about 50 cell generations in dividing cells or for at least about 8 weeks in non-dividing cells under nonselective conditions without an appreciable loss of copy number.
2. The vector comprising papilloma virus sequences consisting essentially of (A) a minimal origin of replication of a papilloma virus, and (B) a minichromosomal maintenance element of a papilloma virus consisting essentially of multiple E2 binding sites, wherein the distance between the minimal origin of replication and the minichromosomal maintenance element is less than about 1.0 kb, wherein the vector, when present in a mammalian host cell which expresses E1 and E2, persists in the cell for at least about 50 cell generations in dividing cells or for at least about 8 weeks in non-dividing cells under nonselective conditions without an appreciable loss of copy number.
3. The vector comprising papilloma virus sequences consisting essentially of (A) a minimal origin of replication of a papilloma virus, (B) a minichromosomal maintenance element of a papilloma virus consisting essentially of the region of BPV mapping to about positions 7590-7673 wherein the vector, when present in a mammalian host cell which expresses E1 and E2, persists in the cell for at least about 50 cell generations in dividing cells or for at least about 8 weeks in nondividing cells under nonselective conditions without an appreciable loss of copy number.
4. The vector comprising papilloma virus sequences consisting essentially of (A) a minimal origin of replication of a papilloma virus, and (B) a minichromosomal maintenance element of a papilloma virus consisting essentially of (BPV E2 binding sites 6, 7, and 8)x wherein x is 3-6, wherein the vector, when present in a mammalian host cell which expresses E1 and E2, persists in the cell for at least about 50 cell generations in dividing cells or for at least about 8 weeks in nondividing cells under nonselective conditions without an appreciable loss of copy number.
As used herein, the term xe2x80x9cconsisting essentially of means that, with respect to papillomavirus sequences, those sequences which are both necessary and sufficient for long-term vector persistence without an appreciable loss of copy number.
In a preferred embodiment of the invention, a vector of the invention will comprise papillomavirus sequences as well as other sequences relating to expression of a gene of interest. The papillomavirus sequences in the vector will preferably consist essentially of a papillomavirus MME and MO, and thus will not contain papillomavirus sequences that are not required for long-term stable persistence in a eukaryotic host cell. The vectors thus advantageously do not contain papillomavirus sequences which are not both necessary and sufficient for long-term persistence in the episomal state. In addition, the vectors do not contain oncogenic sequences which are present in the papillomavirus genome.
In preferred embodiments, the minichromosomal maintenance element of a papillomavirus is from BPV; the minimal origin of replication of papillomavirus is from BPV; the papillomavirus E1 protein is from BPV; the papillomavirus E2 protein is from BPV.
Preferably, the vector further comprises a gene or genes encoding papillomavirus E2 and/or E1 proteins, and the E2 gene most preferably encodes a mutant form of E2 which is a point mutant that is replication competent but defect in transcriptional activation. As used herein, a xe2x80x9cpoint mutantxe2x80x9d may refer to either a single amino acid change, or several individual amino acid changes (2, 3, 4 etc.) which together confer the desired phenotype. A point mutant may be an amino acid substitution or a deletion or insertion.
One particularly useful form of a vector of the invention is a recombinant vector or vector system for stable persistence of erogenous DNA in a host cell, the vector comprising a minimal origin of replication of a papillomavirus, a minichromosomal maintenance element of a papillomavirus, and one or both of the papillomavirus E1 and E2 genes.
The invention also encompasses a mutant form of a papillomavirus E2 protein wherein the replication function of the protein is competent and the transcriptional activation function of the protein is defective, wherein the mutant form of E2 protein differs from the wild-type E2 in a nucleotide point mutation which translates into an amino acid substitution.
Preferred E2 point mutants are mutated in an alpha helical domain, for example in alpha helix 2 or 3, as defined herein.
Additional preferred E2 point mutants useful according to the invention are R37A, E74A, and D 122A and D143A/R172C.
A particularly striking feature of the invention is that the stable vectors of the invention are not restricted to the host cell specificity of papillomavirus. This release from the natural papillomavirus host cell type restriction has been achieved by removing key genetic elements of the papillomavirus genome from their native context; for example, expression of the papillomavirus genes encoding E1 and E2 proteins is not controlled by the promoters that are native to these genes, but rather the E1 and E2 genes are placed under the control of non-native, i.e., heterologous promoters, which are either functional in a broad range of mammalian cells or tissues or are cell- or tissue-specific.
It is preferred according to the invention that the expressible papillomavirus gene encoding E1 or E2 include a structural gene encoding E1 or E2 operatively associated with regulatory sequences for expression of the structural gene in a host cell. Such regulatory sequences will include a promoter and/or may optionally include an enhancer. The promoter is preferably a promoter that is non-native (i.e., heterologous) to the E1 or E2 structural gene. The promoter is may be functional in more than a single tissue type, i.e., one that is able to initiate transcription in a broad range of tissue types, and therefore unrestricted with respect to its tissue specificity. Alternatively, the promoter may be functionally restricted to a single tissue type, and therefore tissue-specific.
As used herein, tissue-specific and cell-type-restricted both refer to wherein a promoter is operable substantially in the same tissue-type or cell-type.
Preferred promoters comprise one of the thymidine kinase promoter and a strong promoter such as the SRalpha promoter. It is expected that a vector of the invention which includes tissue-specific regulatory elements operatively associated with the E1 and/or E2 genes will be capable of long-term persistence only in those cell types in which the regulatory elements are functional.
In its most useful form, a recombinant vector of the invention will include an expressible gene of interest.
A vector of the invention which contains an expressible gene of interest contains not only a structural gene encoding a protein or RNA of interest, but also is operatively associated with regulatory sequences for expression of the structural gene in a host cell. Such regulatory sequences may include not only a promoter, but also additional regulatory sequences such as an enhancer, splice sites, and poly-adenylation sequences. These regulatory elements that control expression of the structural gene promoter may be regulatory elements that are native to the structural gene (i.e., the control sequences that are naturally associated with these genes in their native environment), or they may be non-native to the structural gene, and therefore heterologous regulatory elements. These elements, particularly the promoter, may be functional in more than a single tissue type or may be functionally restricted to a single tissue.
It is expected that a vector of the invention which includes a tissue-specific regulatory element that is operatively associated with a structural gene of interest will express that gene of interest only in those host cell types in which the regulatory elements are functional (i.e., specific).
It is preferred according to the invention that the host cell type-restricted expression (i.e., tissue-specificity) of the structural gene of interest be coordinated with the tissue-specificity of the regulatory elements operatively associated with the E1 and E2 genes. That is, one may envision that the tissue-specificity of E1, E2, and structural gene of interest is the same. Alternatively, the tissue-specificity of E1 and E2 gene expression may be broader than the tissue-specificity of expression of the gene of interest, resulting in a broad host cell type range for long-term persistence of a vector of the invention, and a more limited host cell type range for expression of the gene of interest. Alternatively, the tissue-specificity of E1 and E2 gene expression may be quite limited (for example, to a single cell type), and the tissue-specificity of expression of the gene of interest broad or unlimited, resulting in a limited host cell type in which a vector of the invention can persist long-term, which in turn is the limiting factor in determining the type of host cell in which the gene of interest is expressed.
In another preferred embodiment of a vector of the invention, the vector also includes a bacterial host cell origin of replication and a gene encoding a selectable marker for preparation of vector DNA in a bacterial host cell.
The invention also features host cells containing the vectors herein described, such host cells being most preferably being eukaryotic, and of mammalian origin, such as of muscle, gut, or brain origin.
The invention also features a method of manufacture of a vector, which method includes culturing a host cell containing a vector described herein. It is particularly preferred that such manufacture occur in a lower eukaryotic cells, e.g., yeast or insect, or prokaryotic cells, e.g., bacterial cells such as E.coli or Salmonella. Therefor, the vector will further include an origin of replication of yeast, insect or bacterial origin, and one or more genes encoding a selectable marker, e.g., a gene encoding kanamycin resistance, for selection of cells containing the vector.
The invention also features a method of obtaining stable expression of a gene of interest in a cell, comprising providing a host cell as described above. The invention also features methods of treating a disease stemming from a genetic defect, comprising administering a therapeutically effective or a prophylactic amount the vector of the invention to a patient afflicted with the disease.
The invention also includes use of a recombinant vector of the invention in the treatment of a disease.
The invention also encompasses a gene delivery system comprising a vector of the invention in combination with a gene delivery vehicle, which may be of viral or non-viral origin.
The invention also encompasses a method of producing a protein or RNA of interest in a host cell or a transgenic animal, comprising culturing a host cell under conditions which permit production of the protein of interest, or providing a transgenic animal which produces the protein, as described herein.
The invention also encompasses a mammalian model of disease, for screening of drugs to treat the disease or for testing of therapeutic or prophylactic regimes, the mammalian model comprising a transgenic animal whose cells contain a vector of the invention.
The invention also encompasses a transgenic animal containing an episomal vector as described herein, the vector encoding a protein of interest.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d refers to an animal, preferably a mammal, which contains in some, but not necessarily all, of its cells an episomal vector, as described herein.
The invention also features kits for providing stable persistence of a vector in a host cell, the kit comprising a vector or a host cell as described herein and packaging materials therefore.
A kit of the invention may also include a mutant E2 protein as described herein or a gene encoding this protein, wherein the E2 mutant is thus provided for stable persistence of a vector in a host cell.
Uses and Advantages of the Invention are as Follows.
The invention is useful in in vivo and ex vivo human gene therapy where correction of inherited or acquired genetic defects is desired. The invention also is useful in vaccination protocols where resistance or immunity to infectious pathogens, such as HIV, Hepatitis C Virus, Hepatitis B virus, Herpes virus, parasitic pathogens such as Tuberculosis and Leishmaniasis, and protozoans such as ameobic dysentery, is desired, or the elimination or induced quiescence of aberrant cells, such as cancer cells, is considered beneficial.
Recombinant vectors of the invention are useful in that they permit persistent expression of a therapeutic gene in both dividing and non-dividing cells; for example, in differentiated cells, such as those in brain, and muscle.
Recombinant vectors of the invention are also useful for high level transient expression in cells where desired, such as for cancer therapy or in vivo vaccination.
Both in vivo and ex vivo gene therapy strategies are possible with this vector system, including stable, multicopy gene maintenance and expression, in haemopoietic and other stem cells, and in the committed and differentiated progeny of these cell types.
For human gene therapy, uses of the recombinant vectors of the invention are not limited in terms of delivery of the vector to a cell. That is, vectors of the invention may be delivered to a cell via non-viral or viral delivery systems. Delivery systems of non-viral origin include those which employ cationic liposomes, where vector size constraints do not limit the nature and number of plasmid vector components. Delivery systems of viral origin include viral particle-producing packaging cell lines as transfection recipients for the above E1/E2/MO/MME-containing plasmids into which viral packaging signals have been engineered, such as those of adenovirus, herpes viruses and papovaviruses.
Recombinant vectors of the invention also are useful in transgenesis, including production of transgenic animals via pronuclear injection, or embryonic stem cell transfection and embryo chimera generation.
Recombinant vectors of the invention also are useful for preparation of cell factories for stable, high level expression of proteins of therapeutic value in cultured mammalian cells.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof and from the appended claims.